Conjugates of compounds from the group of polysaccharides and chemical substances which exhibit therapeutic activity and include amino groups are known. For example, WO 2012/004007 discloses conjugates of hydroxyethylated derivative of starch and various medicaments. WO 03/000738 discloses antibiotic/starch conjugates, where the antibiotic is attached to the reducing end of a polysaccharide through a peptide bond. Attachment is Obtained by oxidation of the starch derivative with I2 on its reducing end, in an alkaline aqueous solution, followed by coupling of the oxidized derivative with the antibiotic in an organic solution. Further, from the international application WO03/15826 a pharmaceutical composition is known for inhibiting metastases or preventing recurrence of a malignant cancer, which comprises as an active principle a polysaccharide with a carboxy group linked to an active substance with anticancer activity through amino acid or peptide comprised of 2 to 8 amino acids. The application WO03/074087 pertains to coupling proteins with a starch-derived modified polysaccharide, with the binding interaction between the modified polysaccharide and the protein comprising a covalent bond resulting from a coupling reaction between a terminal aldehyde group of the modified polysaccharide and a functional group of the protein able to react with said aldehyde group. The invention provides also pharmaceutical formulations which include coupled compounds prepared by coupling and use of said compounds for preventive or therapeutic treatment of humans or animals.
From the international application No, WO 2011/069475 a process is known for the preparation of oxidized hyaluronic acid derivatives and a process for modifying such derivatives. According to the process of that application, hyaluronic acid is oxidized with a specific oxidizing agent TEMPO, to obtain an acid derivative with aldehyde groups. The derivative is then used for linking with amines, diamines, amino acids, peptides and other amino-containing compounds. Such linking is implemented by reductive amination with NaBH3CN, in water or a mixture of water and an organic solvent.
The above-mentioned inventions provide couplings (conjugates) of polysaccharides and various kinds of therapeutic substances, however, any of the solutions was aimed at obtaining polysaccharide nanoparticles. Meanwhile, nanoparticles are at present intensively studied as potential carriers for medicaments, due to a number of novel desired properties [Biodegradable nanoparticles are excellent vehicle for site directed in-vivo delivery of drugs and vaccines, Mahaparto A., Singh K., Journal of Nanobiotechnology, 9, 2011]. Nanoparticles with diameters of about 50 nm to about 200 nm, having suitable surface properties, could circulate for a long time in the blood avoiding elimination by kidneys, liver or spleen filtration (long circulating particles, stealth particles). The surface of such nanoparticles should induce neither a response of the immune system nor aggregation of small plasma proteins—opsonins. In that instance, a surface of hydrogel properties, as created by highly hydrophilic polymers such as polyethylene glycol, polysaccharides, polyvinyl alcohol, is particularly desirable. Polysaccharides are especially desirable due to their frequently natural origin, biodegradability and similarity to substances occurring in the body. Such long-circulating nanoparticles tend to accumulate in areas of tumors or inflammations (passive targeting) [Therapeutic Nanoparticles for Drug Delivery in Cancer, Kwangjae Cho, Xu Wang, Shuming Nie, Zhuo Chen, Dong M. Shin, Clin. Cancer Res., 2008 14; 1310]. The effect is due to the fact that cell membranes of endothelial cells padding the circulatory system are tightly sealed by appropriate proteins and a gap between them is several nanometers wide. In the tumor or inflammation area the gaps are much wider and reach several hundred nanometers. This makes the nanoparticles accumulate in the gaps and “leak” from the circulation into the surrounding afflicted tissue, including the tumor. Such passive accumulation of nanoparticles in the disease afflicted area allows for an increased drug concentration in the areas, enhances efficacy of the treatment and reduces side effects. Additional feature of the nanoparticles is their ability to be surface-modified with suitable proteins, metabolites or antibodies to exhibit active affinity to specific cell types, including tumor cells. This allows delivery of medicaments primarily to the afflicted cells, it is desirable to prepare the surface of nanoparticles from polysaccharides due to the fact that tumor cells exhibit significantly increased demand for glucose (the Warburg effect) which in turn allows obtaining an increased affinity of polysaccharide nanoparticles to tumor cells. Such polysaccharide nanoparticles with a drug would penetrate more efficiently the cancer cells and kill them, and when labeled with fluorescent marker, they become an efficient diagnostic tool. Another important application of nanoparticles is gene therapy. A nanoparticle containing a RNA or DNA fragment is able to penetrate a cell and influence the gene-reading processes occurring in the cell. Hope arises to heal genetic diseases.
There are numerous methods for preparation of nanoparticles, but unfortunately most of them are very complex and require application of drastic conditions (ultrasounds, high temperatures), aggressive chemical compounds, toxic organic solvents or surface-active compounds [Biodegradable nanoparticles are excellent vehicle for site directed in-vivo delivery of drugs and vaccines, Mahaparto A., Singh K., Journal of Nanobiotechnology, 9, 2011]. Nanoparticles for therapeutic use should be non-toxic and most preferably biodegradable. Polysaccharides make a very good material for the preparation of such nanoparticles, due to their biocompatibility and biodegradability [Lemarchand C., R. Gref, P. Couvreur, Polysaccharide-decorated nanoparticles, European Journal of Pharmaceutics and Biopharmaceutics 58, 2004]. However, known methods for the preparation of nanoparticles from polysaccharides by attaching hydrophobic groups are complex and require use of surface-active materials or aggressive chemicals [Nanoparticles of hydrophobically modified dextranes as potential drug carier system, Aumelas A., Serrero A., Durand, E., Dellacherie E., Leonard M., Colloids and Surfaces B, 59, 2007]. Nanoparticles such prepared must be further purified for a long period of time as a consequence of toxic properties of the compounds.